Abstract Opiate addiction is a huge public health threat in the United States currently. There is a real need for new treatments that reduce relapse rates, and a major relapse trigger is exposure to conditioned drug cues. The conditionedresponsetoseekdrugisintimatelytiedtothememoryretrievedbytheseremindercues.However, repeated exposure to these cues in the absence of reward can lead to a diminished conditioned response to seek drug, through a process known as extinction. Extinction is thought to establish an opposing inhibitory memory that suppresses the conditioned response, and has been shown to recruit portions of the prefrontal cortex. In particular, the infralimbic (IL) subregion in rodents exerts this inhibitory control over downstream effectors that would otherwise drive the conditioned response. This is true for a number of conditioned behaviors, including conditioned cocaine seeking, food seeking, and fear. Preliminary data suggest that the inhibitory function of this region is masked, but not lost, after heroin self-administration. This may be due to a primary site of action of heroin at mu opioid receptors (MORs) in IL, as activation of IL-MORs elicits feeding and a general hyper-appetitive state (Baldo 2016). Preliminary data suggest that IL-MORs also regulate the motivation to seek heroin, and is consistent with a theory separate appetitive driver and limiter functions exist within the IL cortex. These opposing functions may be encoded by distinct neuronal ensembles within IL with distinct downstream targets. Two major efferent targets of IL that are known to regulate both food and drug seeking are the lateral hypothalamus (LH) and the nucleus accumbens shell (NAshell). We propose that IL neuronalensemblesprojectingtotheLHdriveheroinseeking,whereasthoseprojectingtotheNAshellinhibit heroin seeking. Indeed, preliminary data show that chemogenetic activation of the IL to NAshell pathway reduces cue-induced reinstatement of heroin seeking, similar to our reported effects in cocaine-seeking animals (Augur et al. 2016). We will use a novel TRAP-DREADD system to tag and reactivate different functional neuronal ensembles in IL that drive versus inhibit heroin seeking, and we will identify their downstream efferent targets by retrograde tracing. We will also use pathway-specific chemogenetics to activate these different IL efferent pathways and establish their functional role in cue-induced heroin relapse. Finally, we will use a novel trans-synaptic trans-DREADD strategy to examine the second-order circuits controllingheroinseeking.TheseexperimentswilldeterminethespecificfunctionalneuralcircuitryofILandits downstream effectors, and establish their role in driving vs limiting heroin seeking. Furthermore, this work will provide new insight into the specific neuroanatomy controlling heroin seeking and the extent to which these limitercircuitscanbeexploitedtoreducerelapse.